1. Field of Invention
This invention relates to a method for biology research specifically to such a method that allows replicate samples of living cells to be evenly and reproducibly deposited by gravity sedimentation within a predetermined area.
2. Discussion of Prior Art
A burgeoning field in biology is the study of how cells move. Scientific disciplines applied to the study of cell movement range from developmental biology, neuroscience, immunology, nerve regeneration, cancer cell metastasis, and others. The mechanisms by which cells attach to surfaces, and the responses triggered by those attachments, demonstrate profound control over a cell's behavior through contact with surface molecules. Laboratory techniques which elicit specific, controllable interactions between a cell and external molecules enable advances in understanding the biochemical and genetic basis of these interactions. Conducting studies on cells in response to the external environment requires the placement of cells at specific locations. The state of the art for studies of cell movement response to external molecules is reviewed below.
Attachment assays. The initial interaction of a cell with its extracellular matrix can be studied using an attachment assay (Walther B T et al 1973; Miki I et al 1993). These techniques involve initial coating of a surface with a substrate, typically a protein. Cells are added and allowed to sediment at 4.degree. C. onto the substrate. Once the cells have sedimented, the assay vehicle is moved to 37.degree. C. for physiological attachment. The duration of the adhesion assay may vary according to the cells being studied and the nature of the substrate. After the cells attach, free cells are washed away and the remaining cells are enumerated. This is accomplished using either direct cell counting under a microscope or other indirect measures of cell number. These include counting of cells labeled previously with some radioactive compound, or other calorimetric or fluorescent endpoints indicative of cell number. There is a linear relationship between the measurement and the number of cells by which the fraction of attached cells can be determined. These assays are single endpoint assays of cell adhesion to substrates. Determining the kinetics of attachment requires multiple replicate experiments being analyzed at different times. The mechanism for attachment can be inferred using blocking antibodies that interfere with either certain receptors on the cell surface or that block ligands on the substrate. The state of the art also includes using molecular genetic techniques to alter the levels of expression of genes believed to be involved in cell attachment. Direct studies of morphological changes in the cells, changes in cell growth properties, or evaluation of biochemical and genetic changes by the cells in response to attachment cannot be addressed using these techniques. Additionally, appropriate control conditions in the experiments using mock-manipulated cells are potentially inadequate to properly assess other changes in the cells that may arise indirectly from the experimental manipulation of the cells. For example, transduction of a gene (or genes) into the cells of interest with vectors adds the gene(s) to the cells, but may also change cell behavior in unrelated and unanticipated ways. No permanent record of the endpoint is available. The inability to study the dynamics of cell interactions with the substrate and the impermanence of the endpoint are limitations of this technique.
Cell migration through a membrane. One measure of cell migration currently in use assesses the quantity of cells crossing a membrane under the influence of chemical attractants. The modified "Boyden chamber" assay (Phelps P and Stanislaw D 1969; Jungi T W 1975; Fedun et al U.S. Pat. No. 5,578,492) has afforded many studies of the influence of different compounds, drugs, and reagents on putative cell motility. In this technique, cells are loaded on one side of a membrane; the membrane has pores of a diameter smaller than the diameter of the cells under investigation. Once cells are loaded on one side of the membrane, the chamber is incubated for periods of time. After expiration of that interval the opposite side of the membrane or the opposite chamber is analyzed for the presence of cells that have crossed the membrane. This technique assesses the final condition of the chamber after a biological process has ensued. Experimental manipulations allowed in this system are only possible prior to the start of the study. For example, the cells could be genetically engineered, or the membrane can be pretreated with different substances. Biological processes occurring while the cells transit through the membrane cannot be directly studied since the cells are not accessible. Signal transduction processes in response to the cell's contact with the membrane or other substances cannot be addressed. These assays are attractive for their convenience in set-up, and because the movement of cells through pores has purported geometric analogies to the invasion process in physiological circumstances. However, movement of cells along any path is inherently a one-dimensional act; the cells relocate from point "A" to point "B." Although the route taken may be nonlinear, the moment-by-moment dynamic is one dimensional. The determination of the endpoint of migration in Boyden chambers or modified Boyden chambers is often a consumable measurement (radioactivity counts, cell counts in a hemacytometer; calorimetric staining of cells); this is not a permanent record. Additionally, the volumes of the chambers used for the Boyden chamber assay are such that relatively large amounts of biochemical regents (antibodies, enzyme blocking agents, gene regulatory control factors, etc.) are necessary for treatments in these systems. Because of the cost of many reagents, this is a substantial limitation in this assay. Because factors added to the target chamber may percolate through the membrane pores, and may even coat the linings of the pores, the Boyden chamber may measure response of the cells to soluble (chemotaxis) as well as to insoluble (haptotaxis) factors.
Micro scale monolayer migration assays. One straightforward approach to study of cell movement has been the monolayer "wound" assay (Zetter B R 1980; DiMuzio P J et al 1995) in which a confluent monolayer of cells is scraped, creating an open space or wound in the cell monolayer. As this space is filled in by the remaining cells, cell motility is assessed. Confounding use of this assay is the recognition that as cells are maintained in monolayer conditions, they elaborate an extracellular matrix, which becomes an uncontrollable variable in studies of how different specific matrix proteins influence cell movement. Additionally, mechanical disruption of cell-to-cell connections, caused by the wounding process, may damage the cells in ways difficult to control or address. This assay also suffers from an inability to automate the measurements. Nor does this strategy lend itself to a screening approach to identify factors that influence cell migration.
Independent derivations of monolayer migration assays have recently been reported. In the first, a cell suspension in molten agarose was used to deposit cells in a defined circle on a substrate (Varani J et al 1978; Barak Y et al 1983; Rupnick M A et al 1988; Milner R et al 1997). The distribution of cells that had emigrated from the initial seeding area is visualized by conventional inverted microscopy, and measurements of the distance traveled are made. Microliter volume pipettors enable small areas of initial seeding of cells to be established from which cell migration is monitored. Since the agarose drop is deposited by hand, success in depositing the cells at a precise, predetermined site is variable. Development of an automated measurement process for tracking cell migration would be difficult using this method. Furthermore, different sources of agarose lead to lot-to-lot variations which could impact both the viscosity of the molten agarose leading to inconsistent settling of cells through the medium, as well as the presence of unknown contaminants which could influence cell behavior. Also, experimental treatments with reagents like drugs, antibodies, or antisense oligonucleotides, would need to readily diffuse through the agarose in order to gain access to the migrating cells. This assumption may be spurious. Maintenance of the agarose in a liquid state also requires temperatures of .gtoreq.42.degree. C. which may be difficult to maintain on a substrate while cells are sedimenting out of the agarose. The smaller the drop of agarose, the more rapid the temperature would cool, leading to cells being suspended in the gelled agarose, rather than dropping onto the surface. A warm surface to ensure that the agarose remained liquid would also lead to rapid drying of the agarose, and osmotic changes to the cells. This approach is technically very demanding as far as control of physical variables is concerned.
A reverse approach, the "under agarose migration assay," uses wells cut into a bed of gelled agarose into which cells are deposited for subsequent movement away from the initial site, traveling under the gelled agarose (Nelson R D et al 1975). Physical restraints imposed by cutting or casting wells in agarose lead to use of large areas of deposited cells. Optical distortion imposed by the residing agarose bed, and the inability to use high power optical instruments to assess subcellular structures or antibody-labeled biomolecules, leave this as a simple gross cell movement measurement system.
Recently, Chicione M R and Silbergeld D L (1995) reported modifications in cell seeding strategies using conventional cell culture cloning rings (0.5-0.7 cm diameter) into which cells were seeded as a circle. A mathematical formula was devised with which to chart changes in cell density at different distances from the initial seeded area of cells. The initial seeded area of cells was relatively large (1 cm.sup.2), and the volumes of media to support the cells for the duration of the assay also became relatively large. Furthermore, the migrating cells at the perimeter of the sedimented circle of cells comprise a very small fraction of the total cells under these conditions.
Lastly, Berens et al (1994) used custom produced glass sedimenting cylinders cut from micropipettes as the conduit through which to deposit cells as a defined circle. The area was small (1 mm across), and the volumes needed for the assay were on the order of 20-50 microliters. The glass cylinders were, however, unstable, and would easily tip over or slide on the substrate to sites that were away from the intended area and lead to unusable data. Because these glass cylinders were cut from manufactured glass tubing, the bore of the sedimenting chamber would vary significantly from cylinder to cylinder. This raised the variability in initial measurements for the migration assay. The glass cylinders have a wide base, which physically contacts the surface onto which the cells deposit. When the cylinders are removed, the close space between the cylinder and the surface creates a strong capillary force, frequently dislodging the attached cells. Despite these limitations, monolayer migration assays demonstrate the utility of studies of cell movement on flat substrates.
Remaining deficiencies in current methods to deposit cells are: lack of reproducibility, mechanical difficulties in use, limited throughput of experiments for screening new agents, inability to study cell-cell interactions, and only limited potential for video microscopy or computerized data collection.
Additional material pertinent to the physical and biological features of the invention: